From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

From the Department of Cardiovascular Surgery, Union Hospital (Z.W., K.Z., Y.L., P.Y., J.W., Y.W., F.L., J.L.) and Department of Biochemistry and Molecular Biology (Y.W.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Cardiology, Central Hospital of Wuhan, Wuhan, China (P.Y.); and Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center for Human Genome Research (Y.Y.) and Laboratory of Cardiovascular Immunology, Key Laboratory of Molecular Targeted Therapies of the Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College (Y.Z.), Huazhong University of Science and Technology, Wuhan, China.

Introduction

Abdominal aortic aneurysm (AAA) is a common disease affecting elderly males, with an incidence of 1.3% in men 45 to 54 years old and 12.5% in men 75 to 84 years old, especially in Western countries.1 Aortic rupture carries a 75% to 90% mortality rate, which is related to aortic diameter.2 Surgical repair is the only efficient treatment for elective patients with large AAAs (aorta diameter ≥5.5 cm). However, based on aneurysm screening programs in the United States, >90% (≈360 000) of aneurysms were small aneurysms (3.0 cm<aorta diameter<5.5 cm) when diagnosed by ultrasound or computerized tomography.3 Surgical treatment is not appropriate for patients with early or small AAAs, and there are still no specific medical therapies for AAA. Therefore, these patients are unable to receive effective treatments, except for the watchful waiting strategy.4,5

AAAs are focal lesions mostly found in the infrarenal aorta, proximal to the aortic bifurcation. However, abundant evidence suggests that the entire vascular tree is anomalous in patients with an AAA.6 Many common risk factors, such as family history, male sex, older age, central obesity, cigarette smoking, hypertension, and abnormal lipid profile, are associated with the formation and progression of AAA.7–9 The pathological progression of AAA includes chronic inflammation, oxidative stress, smooth muscle apoptosis, extracellular matrix degradation, and neovascularization within the adventitia.7,10 Inflammation and matrix degradation in the vasculature are crucial for AAA formation. The inflammatory responses in the vascular wall play a key role in matrix metalloproteinase (MMP) expression and vascular smooth muscle cell (SMC) apoptosis.11,12 The main inflammatory cells, for example, macrophages, mast cells, natural killer cells, T lymphocytes, and B lymphocytes, accumulate in aneurysmal segments.13 CD4+ T cells predominate in human AAA lesions, especially T helper (Th)1 cells.14 However, interleukin (IL)-17A plays a pivotal role in facilitating inflammation during AAA formation. Furthermore, AAA formation is attenuated by IL-17A suppression through mesenchymal stem cell treatment.15 IL-17A, also known as IL-17, is a significant regulator of inflammation and apoptosis in the inflammatory response.16 IL-17A and IL-17F are mainly produced by Th17 cells,17,18 although there are many other sources of IL-17A, such as CD8+ T cells,19 natural killer T cells, γδ T cells, and natural killer cells.20 Because Th17 cells were established as a distinct lineage by Harrington et al and Park et al in 200521,22, Th17 cells and IL-17A have been widely studied in many autoimmune and inflammatory diseases.17 Retinoic acid–related orphan receptor gamma thymus (RORγt) was identified as the master transcription factor regulating the differentiation and expansion of Th17 cells23; thus, antagonizing RORγt may become a novel therapeutic strategy for Th17-related autoimmune and inflammatory diseases.

Digoxin, which is derived from the digitalis plant, is widely used to treat atrial fibrillation, heart failure, and other cardiac pathologies. Surprisingly, recent studies ascertained that digoxin and its derivatives can inhibit Th17 cell differentiation and IL-17A production by selectively antagonizing the activity of RORγt.24,25 In an experimental autoimmune encephalomyelitis model, which is a canonical IL-17A–mediated autoimmune disease model, digoxin conspicuously inhibits IL-17A production, attenuating the severity of experimental autoimmune encephalomyelitis.24

In our previous study, we found that digoxin significantly prolonged allograft survival time in the BALB/c-to-B6 mouse cardiac transplantation model by suppressing Th17 cell differentiation.26 However, it is unknown whether the specific RORγt antagonist could attenuate experimental AAA (EAAA). We hypothesized that antagonizing the activity of RORγt via digoxin treatment would inhibit the progression of EAAA by suppressing Th17 cell differentiation and IL-17A expression. Human aortic sample analysis and 2 different EAAA models were used to test our hypothesis.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

To fully investigate the role of IL-17A–related inflammatory responses during AAA progression, we first examined the human tissues. In addition to AAA specimens, normal aortic tissues were collected as the control group. We analyzed all samples by Western blot analysis and microscopic examination. Compared with the control specimens, IL-17A and RORγt were conspicuously increased in aortic tissues from AAA patients (Figure 1A). By semiquantitative histological analysis, CD4+ T cells, macrophage infiltration, and the neovascularization within the adventitia were assessed on each aortic cross-section. We found that all of these parameter values were significantly increased in the AAA group (Figure IC–IH and IJ–IL in the online-only Data Supplement). In addition, to assess the grade of structural damage, the median elastin and SMC destruction was scored on a histology grading scale from mild (I) to severe (IV). Both medial elastin fragmentation and SMC destruction scores were significantly increased in the AAA group (Figure 1B and 1C and Figure IA, B, and I in the online-only Data Supplement), which confirmed the typical pathological progression of AAA.

Interleukin (IL)-17A and retinoic acid–related orphan receptor gamma thymus (RORγt) expression are increased in human abdominal aortic aneurysms (AAAs). A, The protein expression of IL-17A and RORγt was increased in aortic tissue from AAA patients (n=5 per group) compared with control specimens (n=5). B, C, Medial elastin destruction score was significantly increased in the AAA group (n=5 per group). The aortic sections are from different samples. Data are presented as the mean±SEM. Elastic destruction was analyzed with the Mann–Whitney test, and other data were analyzed with the 2-tailed Student’s t test. ***P<0.001 or **P<0.01 vs the control group. Scale bar in the histological images (magnification, ×200); bar=100 μm. EVG indicates elastica van Gieson staining.

Treatment With Digoxin Reduces the AAA Incidence and Increases the Survival Ratio in the Angiotensin II/APOE Model

Although an EAAA was defined as a ≥50% increase in the aortic diameter or the occurrence of an aortic dissection, we comprehensively analyzed the ultrasound results, visible changes during the aortic harvest (Figure II in the online-only Data Supplement), histological assessment, and digital photographs with scaling (only some samples, data not shown) when we defined an EAAA. However, ultrasound diameter (luminal diameter) measurements were the most important considerations. We found that IL-17A–associated inflammatory responses are involved in the progression of AAAs, and so we developed the angiotensin II (Ang II)/APOE model to test our hypothesis. To our surprise, AAA incidence was dose-dependently reduced after digoxin treatment, and the mouse survival ratio was elevated (Figure 2C and 2D). The aortic diameter (luminal diameter) was measured using ultrasonography on days 0, 7, 14, and 28 after Ang II infusion (Figure 2A and 2B). A ≥50% increase in aortic diameter or the formation of an aortic dissection was defined as an AAA. The data for days 0, 7, 14, 21, and 28 were analyzed. The increase in aortic diameter over the 28 days was significantly reduced in the high-dose group (40 μg/d per mouse) compared with the control group (0.5% dimethyl sulfoxide treatment) and low-dose group (20 μg/d per mouse; 1.30±0.07 versus 1.73±0.12 mm, P<0.01 and 1.52±0.08 mm, P<0.05, respectively). However, there was no significant difference between the control group and low-dose group (P>0.05; Figure 2E). Similar to the aortic diameter, the AAA incidence was also reduced by digoxin treatment (Figure 2C). No AAA formation was found in the sham group. Treatment with digoxin dose-dependently decreased the AAA incidence compared with the control group. In the high-dose group, the AAA incidence was significantly reduced from 70% (control group, 14 of 20) to 35% (7of 20). The AAA incidence in the low-dose group was higher than that in the high-dose group (60%, 12 of 20), but there was no statistical significance between them. In line with the reduced AAA incidence, digoxin treatment notably increased the survival ratio from 70% to 90%, compared with the control group (Figure 2D). Although an obviously rising trend existed, there was no statistically significant difference between the vontrol group and the high-dose group (χ2=1.618, P=0.2034).

Treatment with digoxin reduces abdominal aortic aneurysm (AAA) incidence and increases the survival ratio in the angiotensin (Ang) II/APOE model. A, Typical ultrasound images of the abdominal aorta from each group on day 28 after Ang II infusion. B, Representative photographs showing the visible changes in the Ang II–induced AAA model (indicated by the white arrows). C-E, Effects of digoxin on AAA incidence (C, n=20 per group), survival ratio (D, shown in survival curves, n=20 per group; 70% in the control group, 75% in the low-dose group, 90% in the high-dose group. Although an obviously rising trend existed, there was no statistically significant difference between the control group and the high-dose group), and abdominal aorta diameter (E, n=8–9 per group) in the Ang II/APOE model. F, Digoxin treatment had no effect on Ang II–induced hypertension (n=8 per group). Data on day 0 are shown as the baseline. Data are presented as the percentage or the mean±SEM. The χ2 test, t test, and one-way analysis of variance were used for data analysis. *P<0.05 between the marked groups, #P<0.05 or ##P<0.01 vs the control group, **P<0.01 vs the sham group; ns indicates no significance.

Digoxin Treatment Preserves the Structure of the Aortic Wall in the Ang II/APOE Model

Microscopic examination of aortic sections was performed to identify the mechanisms responsible for the digoxin-induced AAA protective effect. Elastica van Gieson staining and α-SMC actin, CD31 immunohistochemistry demonstrated that the elastin fragmentation (Figure 3A–3D), SMC destruction, and neovascularization within the adventitia (Figure III in the online-only Data Supplement) were significantly attenuated by digoxin treatment. A I–IV scale was used to assess the grade of structural damage (I, no elastin/SMCs degradation or mild degradation; II, moderate; III, moderate to severe; and IV, severe degradation), and we found that the disruption of medial elastic lamellar architecture and SMCs was significantly increased in the control group (P<0.001). However, digoxin treatment resulted in the partial reversal of both medial elastin and SMCs. Compared with the control group, the medial elastin fragmentation and SMC destruction scores were significantly reduced in the high-dose group (P<0.01 and P<0.05, respectively). A significant difference in elastin fragmentation was also observed between the low-dose group and the high-dose group (Figure 3M; P<0.05). Similarly, neovascularization was also reduced by digoxin treatment and in a dose-dependent fashion (Figure III in the online-only Data Supplement).

Treatment with digoxin attenuates CD4+ T cell and macrophage infiltration and preserves the structure of the aortic wall in the angiotensin (Ang) II/APOE model. A-D, M, Microscopic examination of medial elastin and analysis of its destruction score (n=5 per group). E-L, N, O, Microscopic examination of CD4+ T cell, macrophage infiltration, and the semiquantitative histological analysis. Black arrowheads indicate positive characterizations in the immunohistochemical photographs. Five sections are from each of 3 different specimens in the sham group and from each of 5 different specimens in other groups follow the principle of random. Data are presented as the mean±SEM. Elastic destruction was analyzed with the Mann–Whitney test, and other data were analyzed with the 2-tailed Student’s t test. ##P<0.01 vs the control group, #P<0.05 vs the control group, *P<0.05 vs the sham group or between the marked groups, ***P<0.001 or **P<0.01 vs the sham group, respectively. Scale bar in the microscopic photograph (×400; 50 μm). ACS indicates aortic cross-section; and EVG, elastica van Gieson staining.

Semi-quantitative histological analysis of CD4+ T cells and Mac2+ macrophages was also performed to assess inflammatory cell infiltration in the aortic sections. Unexpectedly, we observed that the infiltration of inflammatory cells was noted not only in the adventitia, but also in the medial layer and perivascular adipose tissue. A significant increase in CD4+ T cells and Mac2+ macrophages was observed in the control group compared with the sham group (Figure 3E, 3F, 3I, and 3J), whereas both CD4+ T cells and Mac2+ macrophages were reduced in the digoxin-treated groups; a significant reduce was found both in low-dose and high-dose groups compared with control group (Figure 3G, 3H, 3K, 3L, 3N, and 3O). Moreover, immunofluorescent staining of CD4/IL-17A was also performed to determine the distribution of Th17 cells in the aortic walls. A large number of Th17 cells were seen in the adventitia and adjoining tissue in the control group. Strikingly, only a few Th17 cells were noted in the high-dose group (Figure IV in the online-only Data Supplement). These results suggest that inflammatory cells, such as CD4+ T cells (especially Th17 cells) and Mac2+ macrophages, are involved in Ang II–induced AAA progression. And treatment with digoxin dose-dependently attenuates inflammatory cell infiltration in the Ang II/APOE model.

Digoxin Treatment Suppresses the Protein Expression of Pro-Inflammatory Mediators and MMPs in the Ang II/APOE Model

Based on the above results, we conjectured that digoxin retards AAA progression through immunomodulatory effects. We further investigated the molecular mechanisms of digoxin action. The aortic tissues were harvested 4 weeks after Ang II infusion, and the protein levels of IL-17A, RORγt, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ, regulated on activation, normal T cell expressed and secreted (RANTES), and MMP-2 and MMP-9 were determined by Western blot analysis. Compared with the aortic tissues harvested from the sham group, IL-17A, RORγt, MCP-1, IFN-γ, RANTES, and MMP-2 and MMP-9 were significantly increased in the control group. However, treatment with high-dose digoxin significantly reduced the expression of IL-17A, MCP-1, IFN-γ, RANTES, and MMP-2 and MMP-9 (Figure 4A, 4B, and 4C; P<0.05). The levels of RORγt in the digoxin treatment groups were not significantly different compared with those in the control group (Figure 4B; P>0.05).

During the harvesting of aortic tissue on day 28 after Ang II infusion, we found that most spleens in the control group developed splenomegaly, whereas only a few developed splenomegaly in the digoxin-treated groups. The spleen weight and splenocyte number were also analyzed by a manual measurement. Compared with the control group and the low-dose group, both the spleen weight and the splenocyte number were reduced by high-dose digoxin treatment (Figure V in the online-only Data Supplement). This result indicates that the spleen may be involved in the progression of AAAs. To test the effect of digoxin on Th17 cell differentiation, flow cytometry analysis was performed. Surprisingly, we found that the proportion of Th17 cells was significantly increased in the control group; however, it was notably decreased by digoxin treatment, especially the high-dose group (Figure 4D and 4F). Forkhead box P3 (Foxp3)–expressing regulatory CD4+ T-cells (Tregs) have been reported to have anti-inflammatory properties; in addition to Th17 cells, CD4+ Foxp3+ Treg cells were also investigated in the present study. To our surprise, the percentage of Tregs in splenocytes was obviously reduced in the control group. Unlike Th17 cells, treatment with digoxin (especially in the high-dose treatment) significantly augmented the proportion of Tregs (Figure 4D and 4E).

Digoxin Suppresses EAAA Formation in a Model-Independent Manner

In the Ang II/APOE model, we found that treatment with digoxin significantly reduced the AAA incidence, and we investigated the mechanisms of this effect. The results suggested that digoxin suppressed the progression of AAA by suppressing Th17 cell differentiation and IL-17–related immune responses in the Ang II/APOE model. However, we did not know whether digoxin treatment affected only the Ang II–induced model. Thus, we generated another canonical EAAA model with some improvements (Figure 5A). Surprisingly, the AAA incidence and aortic diameter were both significantly reduced by digoxin treatment (40 μg/d per mouse) (Figure 5B, 5C, and 5D). We found no AAA formation in the sham group (saline perfused with 0.5% dimethyl sulfoxide, n=12). However, 10 out of 14 (71.4%, 1 died from thrombosis on day 4) developed AAA in the control group (porcine pancreatic elastase [PPE], 4 U/mL, 100 mm Hg, 5 minutes, treatment with 0.5% dimethyl sulfoxide). Strikingly, only 5 out of 15 (33.3%) had AAA formation in the digoxin group. Elastica van Gieson staining and CD4 and Mac2 immunohistochemistry were also performed in the same manner as described earlier for histology analysis. Compared with the control group, digoxin treatment significantly attenuated the destruction of the medial elastin layer (Figure 6A–6D). A significant increase in CD4+ T cells and Mac2+ macrophages was observed in the control group compared with the sham group. However, both CD4+ T cells and Mac2+ macrophages were reduced in the digoxin group (Figure 6E–6L). Furthermore, digoxin treatment also significantly reduced the medial SMC destruction score and neovascularization compared with the control group (Figure VI in the online-only Data Supplement).

Digoxin preserves the structure of the aortic wall and attenuates IL-17A–mediated inflammatory responses in the PPE/C57 model. A-C, E-G, I-K, Elastica van Gieson staining (EVG) and CD4 and Mac2 immunohistochemistry. D, H, L, Effect of digoxin on structural preservation of the aorta and inflammatory cell infiltration. (n=5 per group). M, N, Inflammatory cytokines increased in the control group but significantly reduced in the digoxin group. Protein band: lane S, sham group; lane C, control group; lane D, digoxin group(n=4 per group). Black arrowheads indicate positive characterizations in the immunohistochemical photographs. Five sections are from each of 3 different specimens in the sham group, and from each of 5 different specimens in other groups, follow the principle of random. Data are presented as the mean±SEM. Elastic destruction was analyzed with the Mann–Whitney test and 2-tailed Student’s t test was used to compare 2 groups. *P<0.05 vs the sham group, ***P<0.001 or **P<0.01 vs the sham group, #P<0.05 or ##P<0.01 vs the control group, respectively. Scale bar in the microscopic photograph (×400; 50 μm). ACS indicates aortic cross-section; IFN-γ, interferon-γ; IL-17A, interleukin-17A; MCP-1, monocyte chemoattractant protein; and MMP-2, matrix metalloproteinase-2.

In addition to the microscopic assessment, the aortic tissue was harvested 2 weeks after PPE perfusion. Western blotting was used for protein expression of IL-17A, MCP-1, IFN-γ, and MMP-2. As found by Sharma et al,15 IL-17A was significantly increased in the control group compared with the sham group, whereas digoxin treatment significantly reduced the expression of IL-17A, MCP-1, IFN-γ, and MMP-2 (Figure 6M and 6N). These results indicate that the valid inhibitory effect of digoxin on experimental AAA disease is model-independent.

Discussion

Our experimental results demonstrate that both IL-17A and RORγt were significantly elevated in aortic tissue from AAA patients compared with normal aortic samples. These results confirm and expand previous results demonstrating that IL-17A plays a pivotal role in facilitating inflammation during AAA formation.15 Th17 cells, a main source of IL-17A, are regulated by their master transcription factor, RORγt.17,19 Huh et al25 found that digoxin and its derivatives can inhibit Th17 cell differentiation and IL-17A production by selectively antagonizing the activity of RORγt. Here, we found that the protein level of RORγt was increased in AAA lesions. However, little is known about the effect of RORγt on AAA progression. Thus, we assumed that antagonizing RORγt would attenuate the progression of AAA by inhibiting Th17 cell differentiation and IL-17A.

To fully investigate the effect of digoxin on EAAA, we created the Ang II/APOE and PPE/C57 models. Surprisingly, digoxin treatment significantly reduced the incidence of AAA in both models. Furthermore, the expression of IL-17A increased in the 2 models. Additionally, treatment with digoxin significantly suppressed the expression of IL-17A– and IL-17A–related proinflammatory mediators in the aortic tissue and changed the proportion of Th17 cells and Tregs in the splenocytes of mice in the Ang II/APOE model.

AAA, which is a permanent, localized expansion of the abdominal aorta, represents a complicated pathophysiological process. It involves proteolysis, SMC apoptosis, neovascularization, chronic inflammatory reactions, and other effects.27 Surgery is the only effective treatment, and the mortality associated with AAA is still ≈50%.28 Moreover, there are no specific pharmacological therapies to prevent the progression of small AAAs.4 Thus, it is necessary to develop a new, effective strategy of drug treatment.

Inflammation plays a crucial role in AAA formation and progression. Oxidative stress, characterized by cell and tissue damage caused by reactive oxygen species and reactive nitrogen species, is implicit in AAA pathogenesis.29 Infiltrating leukocytes are major sources of MMPs that degrade the structural proteins in the vascular wall, such as collagen, laminin, and elastin.30,31 Furthermore, immune cell infiltration can accelerate tissue damage through the release of cytokines (eg, IL-6, MCP-1, and osteopontin), and more immune cells will be recruited to the AAA lesion to induce the apoptotic pathways that involve Fas and perforin. These pathways can lead to the apoptosis of SMCs, which are largely responsible for producing the aortic extracellular matrix.32 IL-17A plays a crucial role in the promotion of vascular inflammation and atherosclerosis.18,33 Pietrowski et al34 reported that IL-17A induces the NAD(P)H-oxidase–dependent production of superoxide and hydrogen peroxide and induces oxidative stress by p38MAPK signaling. Our results from the human aortic samples confirm the pathophysiological process of AAA. In addition to the protein expression of IL-17A and RORγt, medial elastin and SMC destruction scores were significantly increased in the AAA group compared with the control group. Moreover, large quantities of CD4+ T cells and macrophages were noted in AAA samples but not in normal aortic tissue.

As a major effector of the renin–angiotensin system, Ang II is an effective inducer of vascular inflammation. There are 2 important signaling networks involved in AAAs, namely, 1 mediated by the transforming growth factor (TGF)-β–Smad2 pathway, controlling myofibroblast differentiation and T lymphocyte differentiation, and the other mediated by the NF-κB–IL-6 pathway, controlling monocyte activation. IL-6 signaling also promotes the recruitment of Th17 cells, which are necessary for the progression of Ang II–induced aortic inflammation and dissection by promoting macrophage recruitment to the vascular wall.35 In vascular smooth muscle cells, Ang II is a potent inducer of related cytokines, such as MMP-2, MCP-1, and IL-6. However, in in vitro vascular smooth muscle cells culture, we found that the protein levels of IL-6, MCP-1, and MMP-2 were unaffected by digoxin (10 μM) after Ang II (0.1 μM) stimulation (data not shown). Because the Ang II/APOE model exhibits medial elastin degeneration, an inflammatory reaction, thrombus formation, and atherosclerosis, all of which exist in human AAA,36,37 we chose this model as a predominant approach to test our hypothesis.

In our previous study, we found that digoxin treatment, by antagonizing RORγt activity, attenuated acute cardiac allograft rejection in the mouse cardiac transplantation model, and in vitro treatment with digoxin inhibited the process of IL-6–mediated conversion of Tregs into Th17 cells.26 Two different drug doses were used in this study to determine which dose was more effective and whether the effects of digoxin on AAA were dose-dependent.

Surprisingly, we found that continuous infusion with Ang II in ApoE−/− mice induced AAA formation efficaciously. Fourteen out of 20 mice developed AAAs, but the AAA incidence was reduced to 60% and 35% by low-dose and high-dose digoxin treatment, respectively. The survival ratio was also increased by digoxin treatment in a dose-dependent manner. High-dose treatment with digoxin significantly reduced the aortic diameter compared with vehicle and low-dose digoxin treatment. We also found that antagonizing the activity of RORγt by digoxin obviously reduced CD4+ T cell (including Th17 cells) and macrophage accumulation in AAA lesions. In accordance with the histological analysis, the protein levels of inflammatory cytokines, such as IL-17A, MCP-1, IFN-γ, RANTES, and MMP-2 and MMP-9, were reduced in the digoxin-treated groups. However, the expression of RORγt was not affected by digoxin treatment at either a low or high dose. This result supports the point that digoxin treatment electively inhibits the activity of RORγt, instead of its mRNA and protein expression, which was proposed by Huh et al.25

T cell precursors travel from the bone marrow to the thymus for maturation. Mature naïve CD4+ T cells are then transported to secondary lymphoid organs, such as spleen, lymph nodes, and so on.38 The spleen participates in immune responses; in our previous research, we found that digoxin attenuates splenomegaly and changes the proportion of Th17 cells in splenocytes in the BALB/c-to-B6 mouse cardiac transplantation model.26 In our Ang II/APOE model, we unexpectedly found that treatment with digoxin effectively attenuated the splenomegaly caused by Ang II–induced inflammatory reactions. Additionally, the proportion of Th17 cells in the digoxin-treated groups was reduced compared with that in the control group, especially the high-dose group. Tregs have been reported to have anti-inflammatory properties,39 and so CD4+ Foxp3+ Treg cells were also investigated in the present study. Unlike Th17 cells, the percentage of Tregs in splenocytes was obviously reduced in the control group. However, digoxin (especially in the high-dose treatment) significantly augmented the proportion of Tregs in the Ang II/APOE model. Our findings indicated that the balance of Th17/Treg cells was disturbed by digoxin treatment, and increase of Tregs might be involved in the protection of digoxin treatment in Ang II–induced abdominal aortic aneurysm.

In addition to inflammation, Ang II increased blood pressure to a certain degree. However, it is unlikely that this increase in systolic blood pressure contributed to AAA formation in the Ang II/APOE model.40 Cassis et al41 found that infusions of Ang II and norepinephrine promoted similar increases in blood pressure but had different effects on AAA, as well as that attenuation of Ang II–induced increases in blood pressure had no effect on AAA incidence. In other words, AAA formation is independent of the blood pressure–elevating effect of Ang II in this model. All of these findings indicate that IL-17A was upregulated in the Ang II/APOE model and that digoxin treatment significantly attenuated the progression of AAA by inhibiting Th17 differentiation and IL-17A production by antagonizing the activity of RORγt.

To fully explore our hypothesis, a different mouse EAAA model, the PPE/C57 model (intra-aortic PPE perfusion in C57BL/6J mice), was used. Based on the previous study using this mouse model,42 we made some improvements. First, instead of 0.4 U/mL, the ultimate concentration of PPE was 4 U/mL. Second, because the distal aorta is narrower than the infrarenal aorta, we used the reverse direction of perfusion, that is, from the infrarenal aorta to the distal aortic bifurcation.

PPE-induced AAA in small animals is a standard model for research in vivo. Elastase breaks down the medial elastin layer, which determines the structural properties of the aortic extracellular matrix. It elevates the local expression of MMPs, such as MMP-2, MMP-9, and MMP-12. Perfusion with PPE also induces vascular wall infiltration by inflammatory cells.42 Given the acute inflammatory reaction caused by elastase-induced aortic injury, it is not surprising that the production of proinflammatory cytokines, such as IL-1β, TNF-α, and IL-6, was increased.43 However, we found that IL-17A, MCP-1, IFN-γ, and MMP-2 were also elevated in the PPE/C57 model, which confirms and expands on the previous report.15

Based on the results that AAA formation in the PPE-induced EAAA model was IL-17A–dependent, we investigated the potential effect of digoxin treatment in vivo. A more effective drug dosage was adopted. Interestingly, treatment with digoxin decreased the AAA incidence, inflammatory cell (CD4+ T cells, macrophages) infiltration, and inflammatory cytokine expression in both our Ang II–induced EAAA model and our PPE-induced model. Ten out of 14 mice (71.4%) developed AAA in the control group, whereas only 5 out of 15 (33.3%) developed AAA in the digoxin group.

Nevertheless, the pathophysiologic progress of AAA is complex, and the role of IL-17 in AAA is still controversial. As reported by Sharma et al,15 IL-17 plays a critical role in promoting inflammation during AAA formation. However, in a recent study performed by Ziad Mallat et al, overexpression of suppressor of cytokine signaling 3 in T cells and neutralization of TGF-β activity markedly reduced IL-17 production and increased the aneurysm severity. These results indicated the protective role of IL-17 in the process of AAA.44 Both findings were reasonable and convincing according to their respective studies.

As reported by Seki et al45 in 2003, transgenic suppressor of cytokine signaling 3 expression in T cells inhibits Th1 development but promotes Th2 development. Additionally, Yu Wang et al46 reported that TGF-β activity protects against inflammatory aortic aneurysm progression and complications in Ang II–induced EAAA model. Furthermore, a meeting abstract47 also indicates the potential protective effect of TGF-β. Ultimately, in addition to IL-17/Th17 cells, Th2 cells, TGF-β might all be involved in the progression of AAA. To elucidate this mechanism, further studies are necessary.

In summary, our study shows that digoxin, a selective antagonist of RORγt activity, is extraordinarily efficacious in suppressing the formation and progression of EAAA in a model-independent manner. Th17 cell- and IL-17A–related inflammatory responses play a critical role in AAAs. Furthermore, selectively antagonizing the activity of RORγt with digoxin significantly attenuated Th17 cell differentiation and IL-17A–related inflammatory reactions, preserved the structure of the vascular wall during AAA development, and reduced the AAA incidence. Moreover, treatment with digoxin electively inhibited the activity of RORγt but had no effect on the protein expression of RORγt.

Our present research corroborates for the first time that antagonizing the activity of RORγt with digoxin could suppress EAAA progression. However, ≥3 questions are still unresolved. First, the pathophysiologic progress of AAA is complex, and our findings indicate that Th17/IL-17A plays a pivotal role in facilitating inflammation during AAA formation. However, increases of Th17/IL-17A could be a consequence, rather than a cause of Ang II/APOE model, and further studies are warranted. Second, we found that treatment with digoxin could not completely protect the aortic wall against inflammatory injury. Because digoxin has no effect on the expression of RORγt-independent Th17-related moderators, such as IL-21, cMaf, RORα, Batf, and IRF425, digoxin selectively inhibited RORγ- and RORγt- but not RORα-mediated Th17 cell differentiation and IL-17A production. The protein level of IL-17A in the digoxin-treated mice was still higher than that in the sham group. Third, the concentration of digoxin needed to inhibit RORγt-mediated IL-17A production was no <10 μM. However, the toxic concentration for human cells is 300 nmol/L, far less than the experimental concentration we performed. Although digoxin cannot yet be administered to small AAA patients directly, our research indicates that antagonizing the activity of RORγt may become a novel strategy for nonsurgical AAA treatment. Moreover, a novel derivative of digoxin that is more effective and less toxic may be developed based on its molecular characteristics.

Acknowledgments

We are thankful to Dr Yufeng Yao and Dr Yanzhao Zhou for their excellent technical assistance during the study.

Sources of Funding

This work was supported in part by the National Natural Science Foundation of China Grant No. 81270322.

American Association for Vascular Surgery and Society for Vascular Surgery. Guidelines for the treatment of abdominal aortic aneurysms. Report of a subcommittee of the Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery.J Vasc Surg. 2003;37:1106–1117.

Significance

Abdominal aortic aneurysm is a common disease affecting elderly males. Recent study has shown that interleukin-17A plays a pivotal role in facilitating inflammation during abdominal aortic aneurysm formation. Moreover, digoxin and its derivatives can inhibit T helper (Th)17 cell differentiation and interleukin-17A production by selectively antagonizing the activity of retinoic acid–related orphan receptor gamma thymus (the master transcription factor regulating the differentiation and expansion of Th17 cells). We show here for the first time that treatment with digoxin inhibits experimental abdominal aortic aneurysm progression both in Angtensin II/APOE model and porcine pancreatic elastase/C57 model via inhibiting the Th17/interleukin-17A–related inflammatory responses. These findings suggest that antagonizing retinoic acid–related orphan receptor gamma thymus activity by digoxin and inhibiting the Th17/interleukin-17A–related inflammatory responses may become a novel strategy for nonsurgical abdominal aortic aneurysm treatment.